1. Field of the Invention
The present invention relates to the field of recombinant polypeptide production in plants. The present invention also relates to the production of large-scale amounts of the polypeptide by mixing together plant tissue and recombinant microorganisms under controlled conditions to produce large amounts of transiently expressed recombinant polypeptides.
1. Background
The development of plant cell and tissue culture as a technology that will permit rapid evaluation of heterologous gene expression in plants is highly desirable. It would further be advantageous to rigorously monitor, control and operate bioreactors for plant cell and tissue culture. These bioreactor operational principles are utilized to correlate performance of transient expression systems and test expression of polypeptides such as monoclonal antibodies (MAbs), human growth hormone (hGH), blood transport proteins, including human transferrin (hTR) and so on.
The present application is directed to plant cell and root culture as a platform for protein expression in transgenic plants. Pilot-scale production of plant biomass would be a tremendous asset for plant biotechnology companies, particularly those who are developing transgenic plants as a means of producing biochemicals. The conventional establishment and characterization of a stable transgenic plant line involve a long process that takes two or more years. This development time, however, is too long to fit effectively into pharmaceutical drug discovery programs and is also hindering development of other plant biotechnology efforts such as plant-based production of industrial enzymes and modified polymeric materials (such as polyisoprenes, polyhydroxyalkanoates, collagen, and spider silk). The development of bioreactor-based biomass production to provide a more rapid vehicle for the testing of transgenes expressed in plant tissue and delivery of material for characterization and testing would, thus, be highly advantageous.
Despite industrial efforts, conventional information at the pilot scale for plant cell culture is limited, and scale-up from small (less than 5 liters) to pilot scale (50-500 L) remains an art. At the pilot scale, details are usually absent and little more than a growth curve is presented.
The bioreactor requirements for transient gene expression offer an excellent opportunity to refine plant cell culture growth techniques and conduct a more rigorous evaluation of the scientific basis of these control and operational strategies. As will be described in more detail below, the process involves the growth of plant suspended cells or root tissue within a bioreactor. This tissue acts as the expression host for transient expression of a foreign gene upon DNA introduction, analogous with industrially accepted microbial and insect cell culture systems. The process involves the addition of a live bacterial or viral vector to transfer DNA containing the gene for the desired protein to the cultured plant tissues. The timing of addition in terms of culture growth rate, physiological state, biomass and nutrient levels are important factors to achieving transient gene expression at levels sufficient to permit protein recovery and purification. Systematic operational procedures are validated in terms of biomass growth, oxygen mass transfer/demand and nutrient consumption. Both cell suspension and root cultures, as well as non-cultured mixture of plant tissue, may be used.
Thus, there is a need in the art for a method to recombinantly produce polypeptide at the milligram level sufficient for protein characterization.
The present invention has met the hereinbefore described need.
The present application describes technology related to utilizing cultured plant tissues for the rapid expression of heterologous protein in sufficient quantities to permit isolation, characterization and/or purification. An aspect of the present invention is on transient protein expression in a time frame of several days after the introduction of DNA to the process and does not rely on stable chromosomal integration. Growth of plant tissue culture in pilot-scale bioreactor systems provides the capability of introducing the DNA to tissue that is of a precisely defined physiological state. In addition to controlling the environmental conditions of the reactor, the plant tissue can be genetically altered to provide unique protein processing capabilities.
The invention has particular utility in the area of discovery and development of therapeutic proteins in transgenic plants. Although there has been tremendous strides in transgenic plant development, establishing transgenic plants, which express heterologous protein, is still a very slow process in comparison to alternative protein production platforms such as bacteria, yeast and others. The method of the present application reduces the time frame of obtaining quantitative:amounts of protein from a minimum of several months to a few days. The significance of this timesaving is that it takes place in the critical stage where the decisions need to be made on the eventual production platform. This decision is particularly important for therapeutics due to the drug approval process. Using the inventive technology, time-critical comparisons to alternative production platforms (bacteria, yeast, mammalian culture cell, baculovirus and so on) can be made.
It is an object of the present invention to provide a method for recombinantly and transiently producing a polypeptide in a plant tissue, comprising:
i) providing a plant tissue sample in a bioreactor;
ii) adding a sample of an Agrobacterium containing a nucleotide sequence encoding the polypeptide to the plant tissue sample;
iii) co-culturing the plant tissue sample with the Agrobacterium so that the nucleotide sequence is transferred to the plant;
iv) allowing the plant tissue to transiently express the polypeptide; and
v) separating the polypeptide from the mixture.
According to the invention, the process is directed to large-scale transient expression of a polypeptide of interest. The plant tissue sample may include a plant cell, algal cell suspension culture, or a root culture. The plant may be a dicot or a monocot. In the case of a dicot the plant may include, but is not limited to, tobacco, potato, bean and soybean. In the case of a monocot, the plant may include, but is not limited to, corn.
Further, according to the method of the invention, the Agrobacterium may be Agrobacterium tumefaciens or Agrobacterium rhizogenes. Preferably, the Agrobacterium may be an auxotroph that is deficient in its ability to metabolize amino acids, vitamins, and/or nucleic acid precursors. If it is deficient for example in amino acid metabolism, it is desirable that the amino acid be a low cost amino acid, and not present in the co-culture medium in sufficient amounts to facilitate auxotroph growth without supplementation.
In the method of the invention, the polypeptide of interest may be a protein such as an enzyme, antibody or a therapeutic protein. It may also be a biomaterial or a gene product which is being tested for function (transcriptional factor, signal molecule, receptor, etc.).
The method of the invention further includes monitoring and controlling the bioreactor environment. Some of the factors to be monitored and controlled include, but are not limited to, the pH, optical density, temperature, media nutrient levels, dissolved oxygen levels, and polypeptide expression levels. In a preferred embodiment, the pH may be controlled to about 4.9 to 6.1.
As a preferred embodiment, the Agrobacterium may be an auxotroph and be added to plant culture at about 7 to about 14 days of the plant culture. Alternatively, the Agrobacterium can be an auxotroph and can be added to plant culture at a biomass concentration of about 25 to about 35 g(dry weight)/liter. Also, as a preferred embodiment, the time length of reaction between the plant culture and the Agrobacterium may be about 1 to about 4 days.
The amount of polypeptide expected to be obtained from the method of the invention may be more than about 1 mg from about 10 to 1000 liter volume of cells. It is understood that transient expression conditions determined in volumes of about 50 ml to about 1 liter cells can be considered to be predictive of large scale production reaction conditions in about 10 to about 1,000 liter volume bioreactors. The ability to achieve growth of plant cell cultures in bioreactors to small-scale shake flask culture utilizing the control and operational strategies that are outlined in the examples that follow.
In an alternative embodiment to the invention, an Agrobacterium DNA transfer activator may be added to the mixture of plant culture and Agrobacterium culture. The activator may be acetosyringone, syringaldehyde or other phenolic compounds that interact with the Agrobacterium proteins which detect wounded plants.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.